Autophagy Protects Cells From HCV-Induced Defects in Lipid Metabolism Tiziana Vescovo, Alessandra Romagnoli, Ariel Basulto Perdomo, Marco Corazzari, Fabiola Ciccosanti, Tonino Alonzi, Roberta Nardacci, Giuseppe Ippolito, Marco Tripodi, Carmelo Garcia–Monzon, Oreste Lo Iacono, Mauro Piacentini, Gian Maria Fimia  Gastroenterology  Volume 142, Issue 3, Pages 644-653.e3 (March 2012) DOI: 10.1053/j.gastro.2011.11.033 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Analysis of autophagy levels in liver biopsies of HCV patients. (A) Immunoblotting analysis of total protein extracts from liver biopsies of HCV patients using an anti-LC3 antibody. (B) Signal intensities of LC3-I and -II bands have been measured and used for the correlation analysis with microvesicular steatosis and other clinical parameters (see Table 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was analyzed as a protein loading control. Gastroenterology 2012 142, 644-653.e3DOI: (10.1053/j.gastro.2011.11.033) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Autophagy targets lipids in HCV replicon cells. (A, B) High levels of autophagy in HCV replicon cells. (A) Immunoblotting analysis of LC3 in HuH7, blasticidin-resistant replicon cells (Rep Blast), and Rep Neo cells. HCV nonstructural protein 5A (NS5A) was analyzed to verify the presence of HCV replicon expression. Tubulin was used as loading control. (B) Confocal analysis of LC3-positive dots in HuH7 and HCV replicon cells either by means of a GFP-LC3 reporter (upper panels) or by immunostaining using a specific LC3 antibody (lower panels). (C, D) Colocalization of autophagosomes with cholesterol deposits. (C) Rep Blast and Rep Neo cells expressing GFP-LC3 were fixed and stained with the cholesterol-specific probe Filipin (blue signal). (D) Rep Blast cells were fixed and stained using a specific LC3 antibody (red) and Filipin (blue). Arrows indicate dots where cholesterol and LC3 colocalize. (E) Partial colocalization of autophagosomes with neutral and phospholipids. Red fluorescent protein-LC3–expressing Rep Blast cells were fixed and stained with the specific neutral lipid probe Bodipy 493/503 (green signal). Yellow signals in the merge images (right panel) indicate the colocalization of the 2 molecules. Scale bars = 10 μm. Gastroenterology 2012 142, 644-653.e3DOI: (10.1053/j.gastro.2011.11.033) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Cholesterol synthesis is required for autophagy induction in HCV replicon cells. (A) Reduced LC3 cleavage in mevastatin-treated HCV replicon cells. Protein extracts were prepared from HuH7 and Rep Blast cells treated with mevastatin for 36 h and subjected to immunoblotting to determine LC3 and nonstructural protein 5A (NS5A) protein levels. Tubulin was used as a protein loading control. (B) Reduced number of autophagosomes in mevastatin-treated cells. GFP-LC3–expressing Rep Blast cells were treated with mevastatin for 36 h, fixed, and stained with Filipin (blue signal). Scale bar = 10 μm. Gastroenterology 2012 142, 644-653.e3DOI: (10.1053/j.gastro.2011.11.033) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 Inhibition of autophagy causes a large increase of cholesterol deposits in HCV replicon cells. (A) Down-regulation of Beclin 1 expression in HCV replicon. Blasticidin-resistant replicon cells (Rep Blast cells) were transfected with 2 different small interfering RNA oligonucleotides (siBeclin 1a and siBeclin 1b). Forty-eight hours after transfection, protein extracts were prepared and subjected to immunoblotting to determine Beclin 1, nonstructural protein 5A (NS5A), and LC3 protein levels. Tubulin was used as a protein loading control. HCV replicon levels were also analyzed by real-time polymerase chain reaction (see graph). siCtr, unrelated oligo. (B, C) Increased levels of cholesterol in autophagy-impaired cells. (B) HuH7 and Rep Blast cells, transfected as described in (A), were fixed and stained with Filipin (blue signal) and Bodipy 493/503 (green signal). (C) HuH7 and Rep Blast cells were treated with 5 nM Bafilomycin A for 4 h or left untreated, fixed, and stained with Filipin and Bodipy 493/503 and fluorescence intensity measured by fluorimeter analysis. (D) Autophagy inhibition causes massive vacuolization in HCV replicon cells. GFP-LC3 expressing HuH7 and Rep Blast cells treated as described in (A) and (C) were fixed and analyzed for morphological alterations (phase contrast, left panels) and GFP-LC3 localization (right panels). Scale bar = 10 μm. Gastroenterology 2012 142, 644-653.e3DOI: (10.1053/j.gastro.2011.11.033) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Induction of a cholesterol-targeting autophagy in HCV-infected cells. (A) Huh7.5.1 were either infected with HCV JFH1 at multiplicity of infection of 0.1 or left uninfected. Five days post infection, protein extracts were prepared and subjected to immunoblotting to determine HCV Core and LC3 protein levels. Tubulin was used as a protein loading control. (B) Red fluorescent protein–LC3 expressing HuH7.5.1 cells, infected as described in (A), were fixed 5 days post infection and stained with Filipin (blue signal). Scale bar = 8 μm. (C) Four hours before lysis cells, infected as described in (A), were incubated with 5 nM Bafilomycin A or left untreated. Levels of cholesterol and neutral lipids were quantified by Filipin and Bodipy 493/503 staining and fluorimeter analysis. Gastroenterology 2012 142, 644-653.e3DOI: (10.1053/j.gastro.2011.11.033) Copyright © 2012 AGA Institute Terms and Conditions